Regulation Of SGAs Biosynthesis Of Green Tissues In Aerial Part Of The Potato

Steroidal glycoalkaloids(SGAs)is a kind of important secondary metabolites of plant resistance against pests and pathogens in Solonum tuberosum L.Genetic alteration of potato can improve accumulation of SGAs in the foliage.Hence,it has important theoretical and practical significance to enhance plant resistance and to breed new varieties of potato varieties.the solanidine rhamnosyltransferase(SGT3)gene and ribulose-1,5-bisphoshate carboxylase(Ruisco)promoter were cloned in this research.Furemore,cDNA fragment of sgt3 and rbcS promoter were recombinated into plasmid pCEPSP.And we got an plant plasmid with glyphosate resistance marker that has sgt3 gene driven by a green-tissue promoter rbcS(rbcS:sgt3)..In addition,Longshu 3 and Shepody were transformed by Agrobacterium-mediated protocol.The main contents of research and results are as follows.1.Subcloned genes sgt3,nos and rbcS from plasmid pMDsgt3?pMDnos and potato genomic DNA.2.Bioinformatics analysis showed that the full length cDNA of sgt3 contain a 1518 bp opening reading frame(ORF)encoding 505 amino acids with a calculated Mw of 56.985 kDa and pI of 5.02.Moreover,secondary structure of protein encoded by sgt3 indicated that 41.39% amino acids are arranged in alpha helix,16.04% are arranged in extended strand,8.91% are arranged in beta turn and 33.63% are arranged in random coil.Subcellular localization showed that SGT3 protein do not contains chloroplast transit peptide,mitochondrial peptide and signal peptide sequence.Therefore,we speculate that SGT3 protein is located in cytoplasm.3.The rbcS promoter,sgt3 gene and nos terminator were inserted into the base vector pCEPSP by restriction enzyme digestion,and we got an plant recombinant vector with glyphosate resistance marker that overexpressing sgt3 gene driven by a green-tissue promoter rbcS.4.Using Agrobacterium-mediated protocol obtained twelve glyphosate-tolerant transgenic planets.5.The expression level of sgt3 in the transformed plants was determined by RT-qPCR.The results showed that the relative expression level of sgt3 in the leaf are higher(1.3-2.2 times)than non-transformation planets,and the relative expression of sgt3 in the tubers was stable.6.The content of SGAs in different tissues of potato was detected by colorimetric method.The content of SGAs in the leaves of the transformed seedlings increased by 20%-37%,while the changes of SGAs in the tubers was stable.